timsTOF Pro - The new standard for 4D shotgun proteomics

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Dig deeper and faster into the proteome

Mass spectrometry (MS)-based proteomics has become a powerful technology for the identification and quantification of thousands of proteins. However, the coverage of complete proteomes is still very challenging due to the limited speed, sensitivity and resolution of current mass spectrometers.

The timsTOF Pro uses the Parallel Accumulation Serial Fragmentation (PASEF) acquisition method to provide extremely high speed and sensitivity to reach new depths in shotgun proteomics and phosphoproteomics, using low sample amounts.

Achieve near 100% duty cycle for shotgun proteomics

A near 100% duty cycle for high sensitivity, high speed shotgun proteomics can now be achieved with the dual TIMS technology. The novel design allows for ions to be accumulated in the front section, while ions in the rear section are sequentially released depending on their ion mobility. This process is called Parallel Accumulation Serial Fragmentation, or PASEF.

PASEF: the perfect fit for shotgun proteomics

The timsTOF Pro powered by PASEF offers sequencing speed > 100 Hz without losing sensitivity or resolution. This is achieved by synchronizing the quadrupole isolation mass window with the elution time of the specific peptide packages from the TIMS funnel.

Superior robustness, no cleaning required

Many MS instruments used for shotgun proteomics require biweekly or monthly cleaning when run 24 hours a day on large sample cohorts, and performance degradation is noticeable over even shorter time periods. The superior robustness of the timsTOF Pro means that the instrument can be run 24/7 over many days ot weeks without noticeable loss of sensitivity or other performance metrics.

An open-file data format allows researchers to work directly with the raw data and use industry leading software. MaxQuant has been adapted to manage 4-dimensional (4D) features in the space spanned by retention time, ion mobility, mass, and signal intensity which benefit the identification and quantification of peptides, proteins, and posttranslational modifications. PEAKS Studio combines de novo sequencingwith tradtional databbase searches and is optimized for processing timsTOF raw data.

Interview and presentation given by Prof. Juergen Cox at US HUPO 2019

"We now know that the peptide mixtures are still extremely complex when analyzing them in two dimensions (retention time and m/z). Adding one more dimension should in principle get us a long way ahead. In addition to the additional dimension of separation, the timsTOF Pro gives us extremely high speed and sensitivity to get deeper into the proteome and using less sample material.”

timsTOF Pro – Powered by PASEF

Bruker has introduced the TIMS (Trapped Ion Mobility Spectrometry) technology in 2016 as a revolutionary ion mobility technology, achieving unprecedented levels of ion mobility resolution in an extremely compact device. The second generation of dual TIMS analyzer now enables PASEF (Parallel Accumulation Serial Fragmentation) that is setting new standards of uncompromised speed, sensitivity and resolution for shotgun proteomics.

Outstanding hardware performance

The second generation dual TIMS analyzer is optimized for shotgun proteomics needs. Due to its unique geometry ions are released dependent on their mobility from the second section of the TIMS analyzer while the further incoming ions can be accumulated in parallel in the first part of the TIMS analyzer. With the parallel accumulation technology a duty cycle of nearly 100% is achieved resulting in nearly no ion loss.

In the timsTOF Pro an advanced segmented quadrupole mass filter is used for high ion transmission and isolation efficiency. The quadrupole mass position is synchronized with the elution times of the specific ions from the TIMS analyzer. Due to its ultra-high mass position switching time (< 1 ms) it enables the best performance for the PASEF method.

Schematic of the timsTOF Pro

TIMS

Trapped Ion Mobility Spectrometry (TIMS) is first and foremost a separation technique in gas phase, which resolves sample complexity with an added dimension of separation in addition to HPLC and mass spectrometry, increasing peak capacity and confidence in compound characterization. Equally as important, the TIMS device also serves to accumulate and concentrate ions of a given mass and mobility, enabling a unique increase in sensitivity and speed along with the additional dimensinof o separation.

PASEF

With speed in mind, Bruker experts redesigned MS/MS technology to meet the requirements of shotgun proteomics. Peptide ions are separated using trapped ion mobility spectrometry, eluted (~ 100 ms) and detected in the QTOF, generating the TIMS MS heat map (A). In the PASEF method (B) the same TIMS separation is used with the quadrupole isolating a certain ion species during its elution and immediately shifting to the next precursor. Parent and fragment spectra are aligned by mobility values. With the Parallel Accumulation Serial Fragmentation (PASEF) Technology >100 Hz sequencing speed can be achieved. With the PASEF method the MS/MS spectra quality of the low abundant peptides can be increased by selecting them several times.

Parallel Accumulation Serial Fragmentation (PASEF) cycle time: By synchronizing the quadrupole with the TIMS elution time, an average of 12 precursors can be fragmented within a 100 ms timescale. Intelligent software targets low-level precursors multiple times for PASEF MS/MS fragmentation.

timsTOF Pro - Re-defining proteomics throughput and sensitivity

Speed and sensitivity translating into high proteome coverage

The combined power of trapped ion mobility spectrometry (TIMS) and Parallel Accumulation Serial Fragmentation (PASEF) allows for greater proteome or sub-proteome (e.g. Phosphoproteome) coverage without requiring the large sample amounts (typically 1-4µg of a peptide mixture on column) often used in proteomics.

Superior results can be obtained from less than 200ng sample load, therefore reducing both sample preparation costs and MS maintenance frequency. Using a 90 min gradient length more than 5,400 protein groups can be identified from a typical human cell line lysate. The timsTOF Pro can also deliver reproducible quantitative information from smaller amounts of highly complex mixtures.